Efficient and selective methods for introducing exogenous nucleic acid, including various forms of DNA and RNA, into living cells are important for applications in basic research and in therapeutics. Methods which minimize loss of functionality of the nucleic acid and which are selective for types of mammalian cells are of particular importance. Such methods and the nucleic acid-complexing and cell-targeting reagents used in such methods are important tools in basic research and gene therapy. Methods and reagents for efficient introduction of functional antisense and antigene agents into eukaryotic cells are of particular importance.
A variety of therapeutic methods based on introduction of nucleic acids into cells have been proposed. See, for example, Uhlmann, E. and Peyman, A. (1990) "Antisense Oligonucleotides: A New Therapeutic Principle" Chemical Reviews 90:544-584; Helene, C. and Toulme, J. J. (1990) "Specific Regulation of Gene Expression by Antisense, Sense and Antigene Nucleic Acids" Biochim. Biophys. Acta 1049:99-125; Mirabelli, C. K. et al. (1991) "In Vitro and In Vivo Pharmacologic Activities of Antisense Oligonucleotides" Anti-Cancer Drug Design 6:647-661; Cook, P. D. (1993) "Medicinal Chemistry Strategies for Antisense Research" In Crooke, S. T. and Lebleu, B. (eds.) Antisense Research and Applications, CRC Press, Boca Raton, pages 147-189; and Stull, R. A. and Szoka, F. C. Jr. (1995) "Antigene, Ribozyme and Aptamer Nucleic Acid Drugs: Progress and Prospects" Pharmaceutical Research 12(4):465-483.
The introduction of expressible DNA (deoxyribonucleic acids) or RNA (ribonucleic acids) into cells can result in expression of therapeutic peptides to correct genetic defects or inhibit disease conditions. For example, introduction and expression of tumor suppressor genes or metastasis suppressor genes is proposed for cancer therapy. Nucleic acids can also enhance or inhibit gene expression in cells. For example, antisense agents, which target messenger RNA, and antigene agents, which bind to double-stranded DNA, can inhibit undesirable or harmful expression of genes or inhibit viral infection or proliferation. See, for example, WO 92/10590 "Inhibition of Transcription by Formation of Triple Helixes" (Toole et al.) published Jun. 25, 1992. See also, for example, Stull, R. A. et al. (1992) "Predicting Antisense Oligonucleotide Inhibitory Efficacy" Nucleic Acids Res. 20(13):3501-3508; Stull, R. A. et al. (1991), "A Protocol for Selection of Antisense Target Sequences," Pharm. Res. (NY) 10 Suppl. S56 (Sixth Annual Meeting Am. Ass. Pharm. Scientists Washington, D.C., Nov. 17-21, 1996.)
This invention relates, in part, to inhibition of prostate cancer cells by introduction of triplex-forming oligonucleotides into the cells.
Triplex forming oligonucleotides have recently been developed for therapeutic applications (see, for example, PCT application WO 94/05268). Triplex formation is a site-specific phenomenon in which a single-stranded oligonucleotide forms specific G:G-C and A:A-T bonds, by Hoogsteen hydrogen bonding, with a target site in a gene promoter, thereby preventing the binding of nuclear proteins to the promoter. Preferred triplex target sequences are polypurine-polypyrimidine regions within promoter regions of genes, such as oncogenes or proto-oncogenes, the expression of which promote malignancy.
Antisense and triplex-forming oligonucleotides have been evaluated in several tumor models and in viral-infected cells for therapeutic applications. See: Skorski, T. et al. (1996), "Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice," Blood 88(3):1005-1012); Ensoli, B. et al. (1994), "Block of AIDS-Kaposi's sarcoma (KS) cell growth, angiogenesis, and lesion formation in nude mice by antisense oligonucleotide targeting basis fibroblast growth factor," J. Clin. Invest. 94:1736-1746; Skorski, T. et al. (1995), "Leukemia treatment in severe combined immunodeficiency mice by antisense oligonucleotides targeting cooperating oncogenes," J. Exp. Med. 182:1645-1653; Leonetti, C. et al. (1996), "Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and in mice," JNCI 88(7):419-429; Yazaki, T. et al. (1996), "Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide," Mol. Pharm. 50:236-242; Nesterova, M. and Cho-Chung, Y. S. (1995), "A single-injection protein kinase A-directed antisense treatment to inhibit tumour growth," Nature Med. 1(6):528-533; Cooney, M. et al. (1988) "Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro," Science (Wash. D.C.) 241:456-459; Durland, R. H. et al. (1991) "Binding of triple helix forming oligonucleotides to sites in gene promoters," Biochemistry 30:9246-9255; McShan, W. M. et al. (1992) "Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices," J. Biol. Chem 267:5712-5721.
Some literature reports relate to the use of antisense oligonucleotides for treating prostate cancer. Transfection of the LNCaP prostate cancer cell line with a 21 base ligonucleotide antisense to sulfated glycoprotein-2 (SGP-2) was reported to decrease SGP-2 biosynthesis and increase cell death (Sensibar, J. A. et al. (1995), "Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin)," Cancer Res. 55(11):2431-2437). However, a variable response in nude mice growing subcutaneous xenografts of the prostate cancer PC-3 cell line (ranging from no effect to a single complete response) was reported from intralesional injection of antisense oligonucleotides directed against transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR)(Rubenstein, M. et al. (1996), "Antisense oligonucleotide intralesional therapy for human PC-3 prostate tumors carried in athymic nude mice," J. Surg. Oncol. 62:194-200; Rubenstein, M. et al. (1995), "Antisense oligonucleotide induced growth factor deprivation in PC-3 cells enhances bcl-2 expression," J. Urol. 153(4):270A; Rubenstein, M. (1993), "Histologic evaluation of PC-3 tumors treated with antisense oligonucleotides," J. Urol. 149(4):475A).
The human her-2/neu proto-oncogene also known as c-erbB-2, erbB-2, neu, and her-2 identified in human tumor tissue is homologous to the epidermal growth factor receptor, and possesses an intracellular domain with tyrosine specific kinase activity and an extracellular domain (Sumba, K. et al. (1985), "A v-erbB-related proto-oncogene, c-erbB-2, is distinct from the c-erb-1 epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma," Proc. Natl. Acad. Sci. 82:6497; King, C. R. et al. (1985), "Amplification of a novel v-erbB-related gene in a human mammary carcinoma," Science 229:974; Akiyama, T. et al. (1986), "The product of the human c-erbB-2 gene: A 185-kilodalton glycoprotein with tyrosine kinase activity," Science 232:1644-1646).
Overexpression of the HER-2/NEU protein has been documented in many human malignancies, including breast, ovary, colon, salivary gland, stomach, lung, kidney, bladder, and endometrium; however, the greatest proportion of cases that express HER-2/NEU may be in prostate cancer cells(Myers, R. B. et al. (1994), "Expression of p160.sup.erbB-3 and p185.sup.erbB-2 in prostatic intraepithelial neoplasia and prostatic adenocarcinoma," JNCI 86(15): 1140-1145). Recent studies report that 16% to 80% of prostate cancers overexpress HER-2/NEU at the protein level (Mellon, K. et al. (1992), "p53, c-erbB-2, and the epidermal growth factor receptor in the benign and malignant prostate," J. Urol. 147:496-499; Zhau, H. E. et al. (1992), "Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines," Mol. Carcin. 5:320-327; Ross, J. S. et al. (1993), "Contribution of her-2/neu oncogene expression to tumor grade and DNA content analysis in the prediction of prostatic carcinoma metastasis," Cancer 72(10):3020-3028;Kuhn, E. J. et al. (1993), "Expression of the c-erbB-2 (her-2/neu) oncoprotein in human prostatic carcinoma," J. Urol. 150:1427-1433; Cohen, R. J. et al. (1995), "Immunohistochemical detection of oncogene proteins and neuroendocrine differentiation in different stages of prostate cancer," Pathology 27:229-232).
Experimental transfection of an activated neu oncogene into a non-tumorigenic rat prostate epithelial cell line resulted in acquisition of a tumorigenic phenotype (Sikes, R. A. and Chung, L. W. K. (1992), "Acquisition of a tumorigenic phenotype by a rat ventral prostate epithelial line expressing a transfected activated neu oncogene," Cancer Res. 52:3174-3181). When human prostate cancer cell line PC-3 was similarly transfected, those clones with high copy number showed greatly enhanced metastatic capacity (Zhau, H. Y. E. et al. (1996), "Transfected neu oncogene induces human prostate cancer metastasis," The Prostate 28:73-83). These studies show that her-2/neu can act as a single step transforming agent in benign cells, and that increased expression leads to a more aggressive phenotype in human prostate cancer cells.
The her-2/neu gene is a potential target for oligonucleotide therapy in prostate cancer. Inhibition of translation of the mRNA corresponding to the her-2/neu gene by antisense oligonucleotides in cellular systems has been reported (Bertram, J. et al. (1994), "Reduction of erbB2 gene product in mammary carcinoma cell lines by erbB2 mRNA-specific and tyrosine kinase consensus phosphorothioate antisense oligonucleotides," Biochem. Biophys. Res. Commun. 200:661-667).
A purine-rich (G and A) region of the her-2/neu promoter has been identified as a potential target for inhibition by triplex-forming oligonucleotides (Ebbinghaus S. W. et al. (1993) "Triplex formation inhibits HER-2/neu transcription in vitro," J. Clin. Invest. 92:2433-2439). An oligodeoxyribonucleotide designated HN28ap (human neu 28-mer antiparallel, i.e., reversed in orientation with respect to the genomic target) and having the sequence: EQU 5'-G GGA GGA GGA GGT GGA GGA GGA GGA GGA-3' (SEQ ID NO:1)
was reported to form triplexes with the target site in vitro, while the corresponding parallel oligonucleotide did not. In an in vitro run-off transcription assay her-2/neu transcription was reported to be inhibited by HN28ap in a concentration dependent fashion with substantial inhibition seen at 2.5 .mu.M and complete inhibition at an oligonucleotide concentration of 25 .mu.M.
There appear to be no reports of the effect of HN28ap on her-2/neu message or protein levels in prostate cancer cells either in vitro or in vivo. However, a 28 base oligonucleotide differing from HN28ap by one base (T.fwdarw.C) at position 13, was shown to inhibit her-2/neu transcription/translation in the breast cancer cell line MCF7 (Porumb, H. et al. (1996), "Temporary ex vivo inhibition of the expression of the human oncogene HER2 (NEU) by a triple helix-forming oligonucleotide," Cancer Res. 56:515-522). In this case, liposome-mediated transfection was used to introduce the oligonucleotide, free oligonucleotide was not effective, and the effect observed was transient. There has been no definitive demonstration of the effectiveness of oligonucleotide therapy in prostate cancer.
High efficiency introduction of the nucleic acid into living cells is an important aspect of the development of any effective nucleic acid therapeutic. In addition introduction of the nucleic acid therapeutic in to a particular type of cell can be important. For example, specific cell targeting of such therapeutics is critical in cancer therapy directed toward killing cancerous or metastatic cells in order to maximize target cell death and minimize damage to healthy cells. Furthermore, cell targeting of the nucleic acid can result in higher concentrations at the target site and improved therapeutic efficiency.
Several methods that are reported to provide high efficiency introduction of nucleic acids into living cells employ polycations as components of cell transfection compositions. For example, receptor-mediated endocytosis employing polycationic nucleic binding agents covalently linked to ligands of cell surface receptors has resulted in cell-selective, high-efficiency transfection.
Polylysine in its polycationic form binds to polynucleic acids, such as DNA, to form soluble complexes. Polylysine has been employed as a component of compositions for introduction of DNA into cells. Conjugates of polylysine and N-glutaryl-phosphatidylethanolamine (NGPE) effect DNA transfection of cultured cells. These conjugates, carrying an average of 2 NGPEs per polymer, have been designated "lipopolylysine" (Zhou, X. et al. (1991) Biochim. Biophysica Acta 1065:8-14). Lipopolylysine-DNA complexes are believed to bind to cell surfaces and to be internalized into cells via endosomes or lysosomes.
Polylysine covalently linked to a cell receptor specific ligand has been employed to complex DNA and facilitate its entry into cells via receptor-mediated endocytosis. See Cotten, M. et al. (1993) Methods in Enzymol. 217:618-644 for a review of methods relying on receptor-mediated endocytosis. For example, polylysine conjugates with the iron transport protein transferrin are reported to provide highly efficient nucleic acid delivery into certain cells (Wagner, F. et al. (1990) Biochemistry 87:3410-3414; Cotten, M. et al. (1990) Biochemistry 87:4033-4037; and Zenke, M. et al. (1990) Proc. Natl. Acad. Sci. USA 87:3655-3659).
Liver cells (hepatocytes and hepatoma cells, for example) express specific surface receptors for asialoglycoproteins, such as asialoorosomucoid. A soluble DNA carrier and targeting system consisting of an asialoglycoprotein linked to polylysine has been used to bind DNA and hepatitis B virus DNA constructs to liver cells and to effect DNA delivery into targeted cells. Asialoorosomucoid covalently linked to polylysine and complexed to DNA creates a soluble DNA delivery system targeted for cells expressing the asialoglycoprotein receptor. The system has been demonstrated to be receptor-mediated and selective for cells which express the asialoglycoprotein receptor (Wu, G. Y. and Wu, C. H. (1987) J. Biol. Chem. 262:4429-4432; Liang, T. J. et al. (1993) J. Clin. Invest. 91:1241-1246; and Chowdhury, N. R. et al. (1993) J. Biol. Chem. 268:11265-11271).
PCT application WO 91/17773, published Nov. 28, 1991, relates to a system for transporting nucleic acids which displays specificity for T-cells. This system employs proteins capable of binding to cell surface receptors expressed on T-cells, for example proteins capable of binding to CD4, the receptor used by the HIV virus. A polycation conjugate with a CD4-binding protein is complexed with DNA and the resulting complex used to transfer the DNA into T-cells.
A problem with receptor-mediated endocytosis has been the destruction of the nucleic acid by lysosomal action during endocytosis. Addition of replication-deficient adenovirus during transfection via receptor-mediated endocytosis has been reported to enhance delivery of functional DNA into cells. Polylysine-DNA complexes coupled to adenovirus are reported to result in efficient transfer of DNA into cells having adenovirus receptors. Ternary complexes composed of polylysine-adenovirus conjugates and polylysine-transferrin conjugates and DNA are also reported to give efficient DNA delivery (Curiel, D. T. et al. (1992) Human Gene Therapy 3:147-154; Curiel. D. T. et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850-8854; PCT application PCT/EP92/02234, claiming priority to U.S. patent application Ser. No. 07/937,788 filed Sep. 2, 1992; Gao, L. et al. (1993) Human Gene Therapy 4:17-24; and Michell, S. L. et al. (1993) J. Biol. Chem. 268:6866-6869).
Steroid hormones interact with components of biological membranes and may enter their respective target cells by diffusion or by a membrane-mediated process which is saturable and temperature-dependent (Pietras, R. J. and Szego, C. M. (1977) Nature 265:69-72). Plasma membranes of target cells may contain steroid receptors (Steinsapir, J. and Muldoon, T. G. (1991) Steroids 56:66-71). The human androgen receptor (See; Coffey, O. S. (1992) In Campbell's Urology, 6th Edition, Walsh, P. C. et al. (eds.), W. B. Saunders Co., Philadelphia, Pa.) can be found in prostate tissue, metastatic prostate cancer tissue, hair follicles, muscle and skin; it can be cytoplasmic but can also be located in cell membranes. A specific mechanism, associated with the cell membrane, must transport steroids into the target cell before they can bind to the cytosolic receptor (Harrison, R. W. et al. (1974) Biochem. Biophys. Res. Comm. 61:1262-1267).
Androgen receptors have affinity for a number of androgens and other steroids. Prostate tumor cells have affinity for progestagenic and estrogenic steroids and antiandrogens, such as cyproterone (Veldscholte, J. et al. (1990) Biochim. Biophys. Acta. 1052:187-194; and Veldscholte, J. et al. (1992) J. Steroid Biochem. Mol. Biol. 41:665-669).
Pending U.S. patent application Ser. No. 08/283,238, of Petros and Liotta filed Jul. 29, 1994, which is incorporated in its entirely by reference herein, provides a water-soluble system for nucleic acid delivery to cells having androgen receptors. A steroid moiety capable of binding to an androgen receptor is covalently linked to a polycation, such as polylysine, polyarginine, polyhistidine or protamine. The polycation-steroid conjugate is complexed with a single-stranded nucleic acid, such as a single-stranded DNA molecule, and contacted with target cells to effect transfection of the cells with the nucleic acid. Polycations that bind nucleic acids which are targeted to specific cells by covalent linkage to ligands of cell surface receptors can be used to detect the presence and location of cells which express that receptor. The polycation can be labelled for detection by complexation to radiolabelled nucleic acid, for example.
This invention relates, in part, to the use of polycationic oligomers to bind and effect charge neutralization of oligonucleotides. Polycationic oligomers of this invention include certain sequential (or alternating) oligomers with cationic groups, such as lysine (Lys) or arginine (Arg), alternating with non-cationic groups along the oligomer chain. Sequential polycationic oligomers of this invention include polypeptides, e.g. (Lys-AAA).sub.n, where AAA is an amino acid with a non-cationic side group and n is the number of repeating units in the oligomer, oligomers having alternating cationic amino acids and lactate residues, e.g. the sequential copolymer [Lys-Lac].sub.n, and peptoids with alternating cationic groups.
Conformational studies of sequential peptides and DNA complexes with certain sequential peptides have been reported (Kubota, S. et al. (1983) Biopolymers 23:2219-2236 (1) and Kubota S. et al. (1983) Biopolymers 23:2237-2252 (2)). These references report the investigation of the conformation of several sequential polypeptides in surfactant solution including [Lys-Ala].sub.n (n&gt;54); [Lys-Leu].sub.n (n&gt;28); [Lys-Ser].sub.n (n&gt;30); and [Lys-Gly].sub.n (n&gt;34). Methods of synthesis of the listed sequential polypeptides were also reported.
Vives, J. et al (1985) Biopolymers 24:1801-1808 report X-ray diffraction studies of the structure of solid films of several alternating polypeptides including poly(Ala-Lys), poly(Leu-Lys), poly(Val-Lys), and poly(Arg-Leu). The size of these polypeptides was estimated to range from 25-80 amino acid residues. The authors refer to Brach, A. and Caille, A. (1978) Int. J. Pept. Protein Res. 11:128-139 and Barbier, B. et al. (1984) Biopolymers 23:2299-2310 for methods of synthesis. Malon, P. et al. (1988) Peptides pgs. 516-518 report studies of conformational changes induced by interaction with porphyrin in basic sequential polypeptides including (Lys-Ala).sub.n (MWt. range 6,500 to 10,000). Azorin, F. et al. (1985) J. Mol. Biol. 185:371-387 reports X-ray diffraction studies of fibers of DNA complexes with several sequential polypeptides including poly(Leu-Lys), poly(Val-Lys), and poly(Ala-Lys). The authors refer to Brach and Caille (1978) supra and Vives et al. (1985) supra for methods of synthesis of sequential polypeptides.
Barbier, B. and Brach, A. (1988) J. Amer. Chem. Soc. 110:6880-6882 and Barbier, B. and Brach, A. (1992) J. Amer. Chem. Soc. 114:3511-3515 report that certain sequential basic polypeptides can accelerate hydrolysis of oligoribonucleotides under basic conditions (pH 8). Poly(Leu-Lys), poly(Arg-Leu) and poly(Ala-Lys) were reported to hydrolyze 85%, 68% and 33%, respectively, of the phosphodiester bonds of oligo(A)'s over 7 days (at 50.degree. C.) compared to 27% hydrolysis by polylysine under similar conditions. The authors explain the enhanced hydrolytic activity of these basic polypeptides as a result of the spatial geometry of the peptide backbone in a .beta.-sheet conformation and the regular spacing of charged residues (6.9.ANG.) in the sequential peptide comparable to the spacing (6.2.ANG.) between consecutive phosphate groups in the oligoribonucleotide.
Poly(lactic acid) is a biodegradable polymer widely used as a biomedical material. The random copolymer, poly(lactic acid-co-lysine), has been prepared and suggested as an alternative to poly(lactic acid) in biomedical applications, particularly as a matrix material for tissue engineering. See: D. A. Barrera et al. (1993) J. Am. Chem. Soc. 115:11010-11011 and D. A. Barrera et al. (1995) Macromolecules 28:425-432. Incorporation of lysine residues into poly(lactic acid) provides chemically reactive sites for derivatizing the material to alter its surface with biologically active moieties. The first copolymer prepared was reported to have an average molecular weight of 64,000 g/mol and to contain 1.3 mol % lysine. Copolymers ranging in molecular weight from about 7,000 to 95,000 and lysine mol % from 2.4 to 6.4 were also reported. Materials with properties desirable for biomedical applications are reported to contain 1-10 mol % lysine. Higher concentrations of lysine in the co-polymer are said to alter the physical characteristics of the polymer poly(lactic acid) and to be deleterious to degradability. It was also reported that the copolymer was derivatized via its lysine residues with a cell adhesion promoting peptide.
Peptoids, i.e., N-substituted oligoglycines, have recently been considered for the development of pharmaceuticals. H. Kessler (1993) Angew. Chem. Int. Ed. Engl. 32(4):543-544. Peptoid libraries, prepared by combinatorial synthesis and containing large numbers of structurally different peptoids, have been screened for peptoids having biological function. Such libraries have, for example, been screened for peptoids with affinity for binding to ligands (J. A. W. Kruijtzer and R. M. J. Liskamp 91995) Tet. Letts. 36(38):6969-6972; R. N. Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; R. J. Simon et al. (1994) Techniques Protein Chem. V (Academic Press) pp:533-539). A strategy for rational design of peptoids that bind as ligands to proteins has been described (D. C. Howell et al. (1994) Drug Design and Discovery 12:63-75). WO91/19735 (P. A. Bartlett et al.) published Dec. 26, 1991, describes a method for generating and screening peptoid libraries to isolate peptoids with selected biological functions. This published application, which is incorporated in its entirety by reference herein, also describes or refers to literature methods for the synthesis of peptoids, for example by solid phase synthesis, and describes sources of N-substituted glycine analogues as starting materials for peptoid synthesis. The application specifically describes screening of peptoid libraries for peptoids that bind to protein or peptide receptors. Conjugates of selected peptoids to pharmaceutically active compounds, in particular peptoid-trimethoprim conjugates, are disclosed. The application notes that steroid conjugates of peptoids can also be made.